CN110846231B - Laccase-producing fungus mucormycoma hirsutum La-7 and application thereof - Google Patents
Laccase-producing fungus mucormycoma hirsutum La-7 and application thereof Download PDFInfo
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- CN110846231B CN110846231B CN201911220322.6A CN201911220322A CN110846231B CN 110846231 B CN110846231 B CN 110846231B CN 201911220322 A CN201911220322 A CN 201911220322A CN 110846231 B CN110846231 B CN 110846231B
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- laccase
- trametes
- estradiol
- hirsuta
- trametes hirsuta
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Abstract
The invention discloses a laccase-producing fungus named as trametes versicolor (Trametes hirsuta) La-7, deposited in China general microbiological culture Collection center in 27.09.2019 with the culture preservation number of CGMCC No5.2199 and the preservation address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North. Using the fungus Sphaerotheca fuliginea of the present invention (Trametes hirsuta) The extracellular crude laccase produced by La-7 fermentation can effectively remove 17 in natural water and livestock and poultry manureβ-estradiol.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a laccase-producing fungus mucormycosis La-7 and application thereof.
Background
Steroid estrogen in the environment mainly comes from industrial wastewater, livestock and poultry waste, domestic garbage and the like, and can be released into natural water and farmland soil, so that health risks of wild animals and human groups are caused. The steroid estrogens enter animals and humans and disrupt the normal regulatory role of the estrogen in the organism by mimicking, interfering with, or opposing the synthesis, transport, metabolism, and release of endocrine substances in the organism, resulting in endocrine dysregulation and derangement of the organism. In recent years, researchers have frequently detected steroid estrogens such as estrone, 17 β -estradiol, and 17 α -ethinyl estradiol in surface water, groundwater, and farmland soil environments in our country. Exposure of steroid estrogens to the ecosystem poses a serious threat to the safety and health of humans and animals. For example, 17 β -estradiol, at a trace concentration, can not only disturb the hormonal balance in the organism, induce feminization of male fish and abnormal reproductive ability, but also cause metabolic disorders in the human body and induce diseases such as cancer. Therefore, how to effectively remove steroid estrogen in the environment has become one of the difficult problems to be solved urgently in the field of pollution remediation.
At present, advanced oxidation technologies such as ozone oxidation, chemical degradation, electrolytic conversion, Fenton's reagent and ultraviolet irradiation are mainly adopted to reduce health risks and environmental effects caused by steroid estrogen. These techniques can effectively reduce the high-concentration polluted steroid estrogen in the environment, but cannot remove the low-concentration polluted steroid estrogen, and can form toxic conversion products to cause secondary pollution to the environment. Therefore, the use of bioremediation techniques to circumvent the risk of contamination of steroid estrogens in the environment is gradually drawing considerable attention from researchers. The laccase-producing fungi are a type of microorganism commonly existing in an ecosystem, and extracellular laccase secreted by the fungi not only can catalyze the oxidation of various organic pollutants, but also can mediate the pollutants to participate in a humification process and increase the reserves of organic carbon and nitrogen in the environment. It is known that laccase secreting fungi are mainly concentrated in basidiomycota, ascomycota and deuteromycota, the most predominant of which are white rot fungi of basidiomycota.
The fungal laccase belongs to blue extracellular copper-rich oxidase, and has a molecular weight of 40-110KD and a sugar ligand accounting for 10% -45% of the whole molecule. The catalytic core of fungal laccases typically contains 4 copper ions (1T 1 Cu, 1T 2 Cu, and 2T 3 Cu), where T2 and T3 Cu together constitute the trinuclear copper cluster site of the laccase. In laccase mediated oxidation reactions, the T1 Cu site first donates 1 electron to the substrate molecule, followed by internal transfer of electrons from the reducing T1 Cu site to the T2 and T3 Cu sites, and finally the 4 electrons of molecular oxygen are reduced to H at the trinuclear copper cluster site2And O. A large number of data prove that the fungal laccase can be used as a green catalystCatalyzing the one-electron oxidation of steroid estrogens in the environment. In the process, single molecular oxygen is reduced into 2 molecules of H2O, 4 substrate molecules are oxidized to form 4 corresponding reactive free radical intermediates. These free radical intermediates are then coupled spontaneously outside the enzymatic reaction site to form a polymeric C-C or C-O-C covalent polymerization product. The formation of the polymerization product significantly reduces the ecotoxicity of the parent compound, which can be removed by subsequent precipitation and filtration processes.
The free radical coupling reaction of the fungal laccase mediated organic pollutants is a novel, green and environment-friendly ecological pollution remediation technology. Contrary to the conventional chemical oxidation and biodegradation ideas, the fungal laccase mediated single-electron oxidation of the steroid estrogen forms a macromolecular coupling product which not only can eliminate the biological toxicity of the steroid estrogen, but also can increase the organic carbon and nitrogen reserves in the environment. In addition, only a single by-product H is formed during the reaction2O, has the advantages of mild reaction conditions, simple operation, economy, environmental protection, no secondary pollution and the like. Therefore, the laccase-producing fungi are separated and screened from the environment, and the laccase is produced by optimizing fermentation culture process parameters of the laccase, so that the method has important environmental significance and wide industrial prospect for simply, efficiently and greenly avoiding the pollution risk of 17 beta-estradiol in natural water and livestock and poultry manure.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to provide a strain of laccase-producing fungus and use extracellular crude laccase produced by the fermentation of the fungus to remove 17 beta-estradiol in natural water and livestock and poultry manure.
The technical scheme of the invention is as follows:
a laccase-producing fungus is named as Trametes hirsuta (La-7), which is preserved in China general microbiological culture Collection center in 27.09.2019 with the strain preservation number of CGMCC No5.2199 and the preservation address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.
Application of Trametes hirsuta (Trametes hirsuta) La-7 in laccase production.
Application of Trametes hirsuta (Trametes hirsuta) La-7 in degradation of 17 beta-estradiol. The extracellular crude laccase produced by the fermentation of Trametes hirsuta La-7 can effectively remove 17 beta-estradiol in natural water and livestock and poultry manure.
A method for producing extracellular crude laccase by using Trametes hirsuta (La-7) comprises the following steps:
1) inoculating trametes hirsuta La-7 into solid culture medium containing 0.05% guaiacol or 0.5% tannin, and standing at 30 deg.C for 7 d; the formula of the solid culture medium is 20 g.L-1Malt extract, 10 g.L-1Glucose, 3 g.L-1KH2PO4And 18 g.L-1Agar, pH 5.2-5.4;
2) cutting the edge of the hypha cultured in the step 1) to obtain a fungus cake with a diameter of 0.6cm, inoculating the fungus cake into 1L of laccase-producing liquid fermentation culture medium, and culturing at pH 3, 30 deg.C and 150 r.min-1Carrying out shake culture for 3 d; the formula of the liquid fermentation medium is 20 g.L-1Malt extract, 10 g.L-1Glucose, 3 g.L-1KH2PO4，0.5mmol·L-1Cu2+；
3) Placing the fermentation liquor obtained in the step 2) at 4 ℃ for 12,000 r.min-1Centrifuging at high speed for 5min to obtain extracellular crude laccase supernatant; adopting 70% saturated ammonium sulfate at 4 deg.C and 150 r.min-1Shaking for 24h to obtain crude laccase.
Compared with the prior art, the invention has the following beneficial effects:
the invention separates and screens a strain of Trametes hirsuta (Trametes hirsuta) La-7(CGMCC No5.2199) which is a fungus producing laccase. The laccase production activity of the trametes hirsuta La-7 is as high as 65.43 U.mL under the condition of optimizing the fermentation culture of laccase production liquid-1. Mixing 5 U.mL-1Adding concentrated crude laccase into 5 mu mol/L-1After the natural water body (pH5) polluted by 17 beta-estradiol is kept stand and incubated for 2 hours at 25 ℃, the removal rate of the 17 beta-estradiol reaches 94.44 percent; at 200. mu.g/kg-1Adding 4mL of 10U/mL into 17 beta-estradiol polluted pig manure (5g of pig manure, 80% of field water holding capacity and pH of 5)-1Concentrating the crude laccase, uniformly stirring once every 12h, and incubating at 25 ℃ for 7d to remove 17 beta-estradiolUp to 91.02%.
The fungus mucor La-7 for producing laccase provided by the invention can be used for producing crude laccase by liquid fermentation. The fermentation process has the advantages of low production cost, simple operation, high laccase yield and the like. The extracellular crude laccase produced by the chaetomium globosum La-7 not only can efficiently remove 17 beta-estradiol in natural water, but also can be directly used for removing 17 beta-estradiol in pig manure. The reaction has the characteristics of mild conditions, high catalytic efficiency, environmental protection, no secondary pollution and the like, and has important environmental significance and wide industrial prospect in the aspects of realizing the treatment of polluted water, the harmless treatment and the resource recycling of livestock and poultry manure and the like.
Biological material preservation information
Trametes hirsuta (La-7) is deposited in China general microbiological culture Collection Center (CCM) with the address of No. 1 Xilu-Beichen, Chaoyang, Beijing, China, and the preservation number of CGMCC No. 5.2199 is 09/27 days in 2019.
Drawings
FIG. 1 shows the colony and hypha morphology of laccases producing fungus Phellinus linteus La-7;
FIG. 2 is a graph showing the effect of pH on the growth of mycelium of Phellinus linteus La-7 and the yield of laccase;
FIG. 3 is a graph showing the effect of fermentation process parameters on the growth of trametes hirsuta La-7 hyphae and laccase yield;
FIG. 4 effect of pH on the removal of 17 β -estradiol from buffer by Tricholoma trichoderma La-7 laccase;
FIG. 5 shows the effect of trametes hirsuta La-7 laccase on the removal of 17 beta-estradiol from natural water;
FIG. 6 shows the effect of trametes lactinea La-7 laccase on the removal of 17 beta-estradiol from livestock and poultry manure.
Detailed Description
Example 1 separation, screening and identification of laccases producing fungus Phellinus linteus La-7
The soil sample polluted by livestock and poultry manure is collected from surface soil (0-10cm) of a sewage outlet of a forward pig-raising professional cooperative society of Ningshan county in Anhui province, and the average concentration of 17 beta-estradiol in the soil is 134.7 mu g/kg-1. 2g of a fresh soil sample is weighed and placed in sterile water containing 0.9% NaCl and shaken vigorously for 1-2min to promote the fungi to be dissociated from the soil. And separating and purifying the culturable fungi in the soil by adopting a plate marking method. Selecting hypha with good growth vigor, inoculating the hypha to a solid culture medium containing 0.05 percent of guaiacol, carrying out inverted culture at 30 ℃ for 4-8 days, if reddish brown appears at the edge of a colony, indicating that laccase is generated, and primarily judging the laccase generation capacity according to the color development degree and the diameter of a color development ring; selecting fungi with high laccase production capacity, inoculating the fungi to a solid culture medium containing 0.5% of tannic acid, screening out strains with brown color change circles, and repeatedly inoculating to obtain laccase-producing fungi with stable color change circles. The formula of the solid culture medium is 20 g.L-1Malt extract, 10 g.L-1Glucose, 3 g.L-1KH2PO4And 18 g.L-1Agar, pH 5.2-5.4.
15 laccase-producing fungi are co-separated and screened from surface soil polluted by pig manure, wherein strain La-7 is inoculated on a solid culture medium containing 0.05% of guaiacol and 0.5% of tannic acid to respectively generate obvious reddish brown and brown coloration circles, the diameter of the coloration circles is linearly related to the inoculation time, and the linear growth rates are respectively 0.88 and 1.02 cm-d-1The result shows that the strain La-7 has laccase production property. The colony of the strain La-7 is white and round, has a dry surface and is provided with a filament and spores, and the vegetative hyphae are embedded into the culture medium and are not easy to pick up (figure 1). Extracting strain La-7 genome DNA by a CTAB method, and carrying out PCR amplification by using a fungus universal primer. The ITS-rDNA sequence of the strain La-7 is compared in a GenBank database (GenBank accession number: MK104139), and the result shows that the sequence similarity of the strain La-7 and a plurality of Trametes hirsuta reaches 100 percent. Combining the morphological characteristics of the strain and ITS-rDNA sequence homology analysis, preliminarily identifying the strain La-7 as the Trichosporon tricholobus. Is named as Trametes hirsuta La-7 and is preserved in China general microbiological culture Collection center with the culture preservation number of CGMCC No5.2199 and the preservation date of 2019, 09 months and 27 days.
Example 2 optimization of culture conditions for liquid fermentation of trametes hirsuta La-7 laccase production
Subpackaging 50mL laccase-producing liquid fermentation medium (pH 3-7) in 150mL triangular flask, inoculating fungus cake with diameter of 0.6cm, 30 deg.C, 150 r.min-1And (5) shaking and culturing for 7 d. Within a set culture time interval, 5mL of fermentation broth is placed at 4 ℃ for 12,000 r.min-1Centrifuging at high speed for 5min to obtain supernatant as crude laccase liquid. Study of pH, temperature, carbon, nitrogen and Cu2+The influence of single factors such as ion concentration on the laccase production capacity of the chaetomium fortune La-7. By oxidation of 1 mmol. L using a UV-visible spectrometer (UV-2550, Shimadzu, Japan)-1Measuring laccase activity change in fermentation broth before and after reaction by using 2, 6-dimethoxyphenol (2,6-DMP), wherein the reaction system comprises 20 mu L of crude laccase liquid and 3.4mL of 10 mmol.L-1Citric acid-phosphate buffer (C-PBS) and 1 mmol. L -12,6-DMP, colorimetric wavelength set at 468 nm. The laccase activity unit is defined as the amount of laccase change, which causes one unit per minute, within the detection wavelength of 468 nm. The formula of the C-PBS buffer solution is 1.9214 g.L-1Citric acid 1.4196 g.L-1Na2HPO4，pH 3.8。
As a result, as shown in FIG. 2, when the initial pH in the medium was 3, the growth of trametes versicolor La-7 was good and it was possible to form mycelium pellets. After 3 days of culture, the activity of the extracellular laccase produced by the strain reaches the maximum value of 16.13 U.mL-1. Temperature (15-45 ℃), carbon source (glucose GL, maltose MA, lactose LA, sucrose SU, fructose FR, soluble starch SS), nitrogen source (malt extract ME, beef extract BE, peptone PE, ammonium nitrate AN, ammonium sulfate AS, potassium nitrate PN, urea UR) and Cu2+Ion concentration (0-5 mmol. multidot.L)-1) The extracellular laccase production of strain La-7 can also be significantly affected (FIG. 3). When 10 g.L of the culture medium is added-1Glucose, 20 g.L-1Malt extract, 3 g.L-1KH2PO4And 0.5 mmol. multidot.L-1Cu2+When the initial pH is adjusted to 3, the temperature is 30 ℃ and the speed is 150 r.min-1After shaking culture for 3 days, the laccase activity in the fermentation liquor is as high as 65.43 U.mL-1. The activity of the extracellular laccase produced by the chaetomium globosum La-7 can still be maintained at 98.6% after the extracellular laccase solution produced by the chaetomium globosum La-7 is stored for 90 days at 4 ℃, and the activity of the extracellular laccase produced by the chaetomium globosum La-7 is proved to be relatively stable.
Example 3 trametes hirsuta La-7 laccase removal buffer 17 beta-estradiol
Placing fermentation liquor of laccase produced by trametes hirsuta La-7 at 4 deg.C for 12,000 r.min-1Centrifuging at high speed for 5min to obtain extracellular crude laccase supernatant. Primarily separating and purifying crude laccase liquid by salting out method, adding 70% saturated ammonium sulfate into crude laccase supernatant, heating at 4 deg.C for 150 r.min-1Shaking for 24h to precipitate crude laccase, and adjusting the enzyme activity of the collected precipitate to 200 U.mL with ultrapure water-1And (4) storing the stock solution serving as the concentrated crude laccase in a refrigerator at 4 ℃ for later use.
The influence of pH on 17 beta-estradiol in a trametes versicolor La-7 laccase removal experiment system is researched by a batch balance test method. The reaction system was constructed in 25mL glass reactors, each containing 5mL of 10 mmol. multidot.L-1C-PBS buffer and 5. mu. mol. L-117 β -estradiol. 5U/mL of the solution was added-1Concentrating the crude laccase, manually shaking for 10s, and standing the reactor at 25 deg.C in dark for incubation. The enzymatic reaction was stopped by the timed addition of an equal volume of chromatographic methanol to the reactor. The quenched enzymatic reaction was filtered through a 0.22 μm filter and the 17 β -estradiol content of the solution was determined by high performance liquid chromatography (Waters HPLC). The HPLC system was equipped with a Waters 600 pump, Waters 2707 autosampler, Waters 2998 photodiode array detector, and an Agilent ZORBAX Eclipse Plus C18 column (4.6 mm. times.150 mm,5 μm). Setting parameters: mobile phase acetonitrile/water 70/30, flow rate 1mL min-1The column temperature is 40 ℃, the sample injection amount is 20 mu L, the detection wavelength is 280nm, and the determination time is 10 min. The result is shown in figure 4, the trametes versicolor La-7 laccase can effectively remove 17 beta-estradiol in the buffer solution under the acidic condition (pH 4-6), and the apparent pseudo-first order kinetic parameter and the half-life period are respectively 0.027-0.055min-1And 25.86-12.67 min. When the pH value is 5, the 17 beta-estradiol removal rate of the laccases of the chaetomium fortunei La-7 within 120min is as high as 99.31 percent.
Example 4 application of trametes hirsuta La-7 laccase in removal of 17 beta-estradiol in natural water body and livestock and poultry manure
Taking pond water of university of agriculture of Anhui province as test water sample (initial pH of 7.9), adjusting pH of the natural water sample to 5 and polluting 5 mu mol. L-117 beta-estradiol. 5U/mL was added to a 20mL sample of water-1Standing and incubating the lachnum chaetomium La-7 laccase at 25 ℃ for 120 min. At set reaction time intervals, the enzymatic reaction was stopped with an equal volume of chromatographic methanol and the residual amount of 17 β -estradiol in the solution was determined by HPLC. Meanwhile, the influence of the catalytic operation times of the laccases of the chaetomium fortunei La-7 on the removal of the 17 beta-estradiol in the natural water body is researched. And taking a water sample added with inactivated trametes versicolor La-7 laccase as a blank control. The effect of trametes versicolor La-7 laccase on the removal of 17 beta-estradiol in natural water is shown in FIG. 5. As can be seen from FIG. 5, as the number of catalytic cycles of laccase was increased from 1 to 3, the 17 β -estradiol removal rate decreased from 94.44% to 78.14% and the half-life increased from 28.52min to 50.97 min. These results indicate that trametes hirsuta La-7 laccase can be used as a green catalyst for removing 17 beta-estradiol in natural water.
Adopting pig manure of a certain farm in the mixed fertilizer market as the manure of livestock and poultry to be tested, freeze-drying and grinding a fresh pig manure sample uniformly, and then polluting 200 mu g/kg-117 β -estradiol. 5g of pig manure sample contaminated by estrogen is weighed, and 4mL of 10 U.mL is added-1The trametes versicolor La-7 laccase (80% of field water holding capacity) is placed in dark at 25 ℃ and is kept stand and incubated for 7d, and the mixture is uniformly stirred once every 12 h. 5mL of a mixture of acetone and methylene chloride (v/v: 1/1) was added to 1g of the freeze-dried pig manure sample at predetermined reaction time intervals, and the mixture was extracted by ultrasonic extraction for 30min to extract 17 β -estradiol. After this step was repeated 3 times, the extracts were combined and reconstituted with 5mL of methanol and passed through a C18 solid phase extraction column. 5mL of the methanol eluate was concentrated to 1mL by a nitrogen blower, and the concentrated solution was subjected to HPLC analysis after passing through a 0.22 μm filter. The group treated with addition of inactivated trametes versicolor La-7 laccase was used as a blank control. The result shows that the removal rate of the trametes hirsuta La-7 laccase to the 17 beta-estradiol in the livestock and poultry manure is up to 91.02 percent after 7d incubation, and the kinetic parameters and the half-life period of the laccase are respectively 0.67d-1And 1.03d (FIG. 6). These results demonstrate that trametes versicolor La-7 laccase can be used for efficiently removing 17 beta-estradiol from livestock and poultry manure.
Claims (4)
1. A laccase-producing fungus is named as Trametes hirsuta (La-7), which is preserved in China general microbiological culture Collection center in 27.09.2019 with the strain preservation number of CGMCC No5.2199 and the preservation address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.
2. Use of Trametes hirsutus (Trametes hirsuta) La-7 according to claim 1 for the production of laccase.
3. Use of Trametes hirsutus (Trametes hirsuta) La-7 according to claim 1 for the degradation of 17 β -estradiol.
4. A method for producing extracellular crude laccase using Trametes hirsuta (Trametes hirsuta) La-7 as defined in claim 1, comprising the steps of:
1) inoculating trametes hirsuta La-7 into solid culture medium containing 0.05% guaiacol or 0.5% tannin, and standing at 30 deg.C for 7 d; the formula of the solid culture medium is 20 g.L-1Malt extract, 10 g.L-1Glucose, 3 g.L-1 KH2PO4And 18 g.L-1Agar, pH 5.2 ‒ 5.4.4;
2) cutting the edge of the hypha cultured in the step 1) to obtain a fungus cake with a diameter of 0.6cm, inoculating the fungus cake into 1L of laccase-producing liquid fermentation medium, and culturing at pH of 3, 30 ℃ and 150 r.min-1Carrying out shake culture for 3 d; the formula of the liquid fermentation medium is 20 g.L-1Malt extract, 10 g.L-1Glucose, 3 g.L-1 KH2PO4，0.5 mmol·L-1 Cu2+；
3) Placing the fermentation liquor obtained in the step 2) at 4 ℃ for 12,000 r.min-1Centrifuging at high speed for 5min to obtain extracellular crude laccase supernatant; 70 percent saturated ammonium sulfate is adopted at 4 ℃ and 150 r.min-1Shaking for 24h to obtain crude laccase.
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